SUMMARY: Celiac disease (CeD) is a gluten-induced, HLA-DQ2 or -DQ8 dependent inflammatory disorder of the small intestine for which no non-dietary therapy is available. The broad goal of this collaboration is to advance our fundamental understanding of CeD pathogenesis with an emphasis on defining the role of extracellular transglutaminase 2 (TG2) and by thioredoxin-1 (TRX) the onset of CeD and the development of villous atrophy. Whereas the pathogenic role of TG2 has been widely discussed in the CeD literature, this hypothesis has never been experimentally tested. Our integrated workplan combines the development of new tools in the Khosla lab with their deployment in reverse engineered mouse models of CeD in the Jabri lab. In addition to prviding key insights into the pathogenesis of CeD, our studies will preclinically validate TG2 and/or TRX as drug targets for treating CeD. The following Specific Aims are proposed: 1) Development of tools to image changes in intestinal TG2 activity: At the recommendation of previous reviewers, we have extensively refocused tool development efforts in two innovative directions. We will: (i) Engineer a ?clickable? probe to visualize the precise locations of catalytically active TG2 in the intestine via post-mortem histological analysis in mice dosed with the probe prior to euthanasia; and (ii) Develop a urinalysis method to detect changes in small intestinal TG2 activity in humans and mice. 2) Study effects of blocking TG2 and TRX on the prevention and reversal of CeD pathogenesis: This Aim is designed to test for the necessity of TG2 and TRX activity in CeD pathogenesis. Gluten-fed HLA-DQ8/Dd-IL- 15/villin-IL15 transgenic mice have all the major hallmarks of CeD, including the appearance of deamidated gluten-specific T cells and antibodies, and intestinal villous atrophy. These triple transgenic mice upregulate TG2 activity and TRX expression in the small intestine. They represent an ideal model to test the role of extracellular TG2 in CeD pathogenesis (for which we have promising preliminary data) as well as the requirement of TRX for its allosteric activation. An analogous triple transgenic mouse strain harboring HLA- DQ2 will be similarly evaluated. Notably, these studies are designed to interrogate the role of TG2 and TRX in the prevention and reversal of CeD, because preventive approaches could be applied to children at high risk of developing CeD, whereas disease-reversing therapies will be useful in adults with established CeD. 3) Investigating the effect of TRX-mediated TG2 activation on CeD pathogenesis: This Aim is designed to establish whether constitutive upregulation of TG2 activity in the intestine is sufficient to induce loss of oral tolerance to gluten and/or the appearance of CeD in gluten-fed mice with appropriate genetic backgrounds. To do so, we will use a newly developed method for up-regulating intestinal TG2 activity in DQ2 and DQ8 transgenic mice using TRX. Because TRX can also endow antigen presenting cells with an inflammatory phenotype, we will determine which of the pathogenic effects mediated by TRX are strictly TG2 dependent.